Acquisition of Philips CM200-FEG, CM120 Electron Microscope and Accessory Equipment for Molecular Microscopy

我们寻求资金建立一个先进分子显微镜中心。所要求的仪器包括一台飞利浦CM200T FEG、一台飞利浦CM120-CRYO、一个加坦低温传输系统、每个显微镜的除污器和干泵站、一个加坦1k x 1k慢扫描电荷耦合器件和一个加坦成像滤光片。选择飞利浦仪器是基于仪器的易用性和已知质量，以及该公司提供的服务的可靠性和质量。新的显微镜和冷冻附件将取代现有的已有8年历史的设备，根据在CM200T-FEG上收集的样本的初步数据，这些设备在性能或分辨率方面不足以获得我们需要的数据，以便对我们的样本进行下一层次的结构理解。根据过去6个月进行的测试，新仪器将极大地提高可以收集的数据的质量和数量。场发射电子枪(FEG)为成像提供了更相干的电子束。因此，与传统图像相比，图像对比度传递函数保持较强的分辨率，从而允许从图像中提取更高分辨率的数据。我们的初步数据表明，在中等分辨率(20-40)和高分辨率(3.5-10)下，CM200T-FEG在我们的标本图像中获得的分辨率都有非常显著的-有时是戏剧性的-改进。这两种显微镜都采用了新的飞利浦“Compu-Stage”，这是一种经过大幅改进的电脑控制测角仪。新测角器的设计(与冷台的低温部分没有接触)导致了更大的样品稳定性。冷级在插入后和级平移后非常迅速地平衡。我们估计，这些优势将使数据收集速度提高5-10倍。用户组的研究重点是三维分子显微镜：通过冷冻电子显微镜和图像分析确定分子和大分子组装体的近原子、S二级、三级和四级结构。用户组的共同目标是计算生物分子和大分子复合体的三维结构，以确定它们是如何工作的。在所有情况下，比我们目前的仪器所能达到的分辨率更高的分辨率是取得进展的关键。正在研究的生物标本包括Acto-myosin、NCD-微管和Kinesin-微管复合体(Milligan)、膜蛋白和离子通道(Milligan、Mitra、Unwin、Yeager)、动植物病毒(Beachy、Johnson、Yeager)和合成有机纳米管(Ghadiri)。培训是活动的一个组成部分。博士后研究员和研究生将接受高分辨率冷冻电子显微镜(成像和电子衍射)、图像分析、高分辨率结构测定和结构解释方面的高级理论和实践培训。拟建的先进分子显微镜中心将包括以下技术：冷冻电子显微镜、200千伏成像和电子衍射、场发射电子源、模拟和数字图像采集以及零能量损失成像和电子衍射。连同用户组中已有的用于图像分析和结构确定的计算和可视化能力，所请求的仪器将创建一个独特的研究和培训设施，将对区域、国家和国际研究基础设施产生重大影响。

We seek funds to establish a center for advanced molecular microscopy. The requested instrumentation includes a Philips CM200T FEG; a Philips CM120-cryo; a Gatan cryo-transfer system, anticontaminator, and dry-pumping station for each of the microscopes; a Gatan 1k x 1k slow-scan CCD and a Gatan Imaging Filter. The choice of Philips instruments is based on ease of use and known quality of the instruments, and the reliability and quality of the service from this company. The new microscopes and cryo-accessories will replace existing 8-year-old equipment which, based on preliminary data on our specimens collected on a CM200T-FEG, is not adequate in terms of performance or resolution for obtaining the data we need to proceed to the next level of structural understanding of our specimens. Based on tests carried out over the last 6 months, the new instruments will dramatically improve both the quality and the quantity of data that can be collected. The field emission electron gun (FEG) provides a more coherent electron beam for imaging. Consequently, the image contrast transfer function remains strong to much higher resolution than on conventional images, thus allowing higher resolution data to be extracted from the images. Our preliminary data indicate that at both moderate (20-40 and high (3.5-10 resolution, the CM200T-FEG provides very significant - sometimes dramatic - improvements in the resolution obtained in images of our specimens. Both microscopes incorporate the new Philips "Compu-Stage" - a much-improved, computercontrolled goniometer. The design of the new goniomenter (there are no contacts with the low temperature part of the cold stage) results in much greater specimen stability. The cold stage equilibrates very rapidly after insertion and after stage translations. We estimate that these advantages will speed up data collection by a factor of 5-10. The research of the user group focuses on 3-D molecular microscopy: determination of the near-atomic, s econdary, tertiary and quaternary structure of molecules and macromolecular assemblies by cryo-electron microscopy and image analysis. The common goal of the user group is to calculate 3-D structures of biological molecules and macromolecular complexes to determine how they work. In all cases, higher resolution than can currently be achieved with our present instrumentation is essential for progress. The biological specimens under investigation include acto-myosin, ncd-microtubule and kinesin-microtubule complexes (Milligan), membrane proteins and ion channels (Milligan, Mitra, Unwin, Yeager), plant and animal viruses (Beachy, Johnson, Yeager), and synthetic organic nanotubes (Ghadiri). Training is an integral part of the activities. Postdoctoral fellows and graduate students will receive advanced theoretical and practical training in high resolution cryo-electron microscopy (imaging and electron diffraction), image analysis, high resolution structure determination and structure interpretation. The proposed center for advanced molecular microscopy will incorporate the following technologies: cryo-electron microscopy, 200kV imaging and electron diffraction, a field emission electron source, analog and digital image acquisition, and zero-energy-loss imaging and electron diffraction. Together with the computational and visualization capabilities for image analysis and structure determination that already exist within the user group, the requested instrumentation will create a unique research and training facility which will have a major impact on the regional, national and international research infrastructure.